Idiopathic Parkinson's Disease (PD) is a major neurodegenerative disease affecting at least 1 million Americans, and the cellular cause of PD is not yet known with certainty. This proposal will explore further the central hypothesis that defects in mitochondrial electron transport chain (ETC) function are a major contributor to premature cell death in PD and will address four Specific Aims 1) define the pathophysiology of mitochondrial transition pore function, and how regulation of membrane potential and intracellular calcium signaling are altered in PD; 2) determine mechanisms of Bcl protein regulation in PD cybrids, and whether transfection with Bcl-overexpression vectors alters mitochondrial function and improves survival; 3) further define the interactions among MAPKinase signaling pathways and NFkappaBeta transcription factor in PD; and 4) characterize mitochondrial transition pore complexes isolated from human postmortem PD brain and compare their function to those isolated from control brain. This project will make use of state-of-the- art intracellular ion imaging technology, RT-PCR techniques, gene transfection strategies, and will develop cell-free systems to examine several inter-related hypotheses. Behind all of these laboratory experiments is a therapeutic imperative, which will be explored in cell and cell-free models. Because new data presented in this application supports the hypothesis of systemically increased oxidative stress in PD patients, exploring these events in an established cell model is even more compelling. This proposal will also compare findings in PD cybrids with those in SY5Y cells exposed to chronic rotenone treatment, a pharmacological cell-based model of complex I loss. Ultimately, the results from this proposal will establish the central importance of genetically acquired mitochondrial ETC dysfunction as an etiologic factor in sporadic PD. Paradigms for evaluating neuroprotective therapies will also be developed to allow targeted approaches to correcting consequences of increased oxidative stress in cells.